Erythropoiesis
Erythropoiesis is the production of red blood cells. Under normal physiological conditions, erythropoiesis is principally regulated by erythropoietin (Epo), a hormone produced by the kidney in response to hypoxia. Erythropoietin, produced by the renal peritubular endothelium, circulates to the bone marrow where it stimulates committed stem cell progeny called erythroid progenitors to produce red blood cells.
Two distinct types of erythroid progenitors have been identified based on their abilities to form morphologically recognizable colonies when grown in semi-solid media such as methylcellulose. The burst forming unit-erythroid (BFU-E) represents the earliest identifiable progenitor fully committed to erythropoiesis. The BFU-E forms large multi-lobular hemoglobinized colonies, possesses a capacity for self-renewal and most (80-90%) are quiescent. BFU-E differentiate to give rise to the colony forming unit-erythroid (CFU-E). The CFU-E is a more differentiated erythroid progenitor which forms smaller hemoglobinized colonies and lacks the capacity of self-renewal. The majority of CFU-E are actively dividing. As BFU-E differentiate into CFU-E there is a loss in the expression of the primitive stem cell surface glycoprotein CD34, and an increase in the expression of the erythropoietin receptor (EpoR) and the transferrin receptor (CD71). Although BFU-E express low numbers of receptors for erythropoietin, they are stimulated by Epo to proliferate and differentiate into CFU-E which, in turn, express higher levels of the Epo receptors.
Erythroid Cell Proliferation and Differentiation
Erythroid proliferation and the differentiation beyond the CFU-E stage is dependent upon erythropoietin and is characterized by the expression of the red blood cell membrane protein glycophorin A, the accumulation of additional erythroid-specific membrane proteins, and the induction of hemoglobin synthesis. The later stages of erythroid differentiation are best characterized by the accumulation of hemoglobin, which accounts for approximately 95% of the protein present in the mature red call. Erythropoietin-stimulated hemoglobin synthesis is normally coordinated within differentiating red cell precursors so that the synthesis of the constituent alpha and beta globin chains is concurrent with the synthesis of heme.
Globin genes, as well as other genes encoding multiple enzymes along the heme synthesis pathway are transactivated by the major erythroid transcription factor, GATA-1, which is expressed following the activation of the Epo receptor by the binding of Epo. Whether Epo will support primarily erythroid differentiation or proliferation appears to depend on the concentration of Epo and the status of the cell cycle. Low concentrations of Epo support xcex2-globin production and prolong the G1 phase of the cell cycle, whereas higher Epo concentrations promote cell proliferation and shorten the G1 phase.
Erythropoietin Receptor
Erythropoietin stimulates erythroid proliferation and differentiation by interacting with a specific receptor expressed almost exclusively on erythroid progenitors. The murine and human EpoR genes and cDNAs have been cloned (D""Andrea et al. (1989) Cell 57:277, Winkelmann et al (1990) Blood 76:24, Jones et al (1990) Blood 76:31). Sequence analysis of the isolates cDNAs revealed that the murine and human EpoRs are 507 and 508 amino acids long respectively, sharing an overall 82% amino acid identity. The topology of the EpoR is such that there is an amino terminal extracellular domain consisting of 226 amino acid (after cleavage of the 24 amino acid signal peptide), a 22 amino acid transmembrane domain and a 236 amino acid intracellular domain. The EpoR is a member of the cytokine receptor superfamily and possesses the characteristic pentapeptide WSXWS motif along with four conserved cysteine residues within the extracellular domain.
The binding of erythropoietin to the EpoR results in the phosphorylation of the intracellular tyrosine kinase, JAK2, which, in turn, phosphorylates several intracellular proteins including STAT5, PI3 kinase and vav. Evidence suggests that activation of second messengers by phosphorylation contributes to the Epo-induced proliferative response; however, the molecular basis which determines whether an erythroid cell will either proliferate or differentiate in response to Epo is unknown.
Characterization of EpoR Mutations
Various mutations have been described which render the murine EpoR either hypersensitive to Epo or constitutively active. Most studies into the functionality of the mutated EpoRs have been conducted using the BaF3 cell line. BaF3 cells are a murine IL-3-dependent pre-B cell line. These cells can be rendered IL-3-independent by over-expressing the EpoR and supplanting murine IL-3(xe2x80x9cmIL-3xe2x80x9d) with human Epo. Using this model, a frame-shift mutation resulting in the replacement of the C-terminal 42 amino acids of EpoR with Ala-Leu was shown to render the murine EpoR hypersensitive (Yoshimura et al (1990) Nature 348:647). This truncated EpoR, when expressed in BaF3 cells, is 3-5 times more responsive to Epo than the wild-type EpoR. It has been demonstrated that this C-terminal truncation removes a negative regulatory domain from the intracellular domain of the EpoR (Klingmuller et al (1995) Cell 80:729-738, D""Andrea (1991) Mol Cel Biol 11:1980). Normally, the hematopoietic protein tyrosine phosphatase SH-PTP1 docks to the C-terminal, dephosphorylating and inactivating JAK2 and thereby decreasing the signalling of the activated EpoR. Removal of this C-terminal negative regulatory domain prevents SH-PTP1 from binding to the EpoR thus resulting in prolonged signalling due to the delayed inactivation of JAK2. Transgenic mice have been generated which express a C-terminal truncated hypersensitive EpoR under the control of the xcex2-actin promoter (Kirby et al (1996) Proc Natl Acad Sci 93:9402). Phenotypically the trangenic mice were normal; however, upon treatment with exogenous Epo there was a marked increase in pluripotent, clonogenic hematopoietic cells (CFU-S) in the transgenic mice as compared to the normal controls. CFU-S are pluripotent hematopoietic progenitors which give rise to granulocytes, erythroid cells, macrophage and megakaryoctes. The number of committed erythroid progenitors (BFU-E and CFU-E) were not significantly different between the transgenic and control mice.
A constitutively active form of the murine EpoR (but not the human EpoR) has also been previously identified. A point mutation whereby Arg129 (position is relative to the putative amino terminus at residue 25), which resides within the extracellular domain of the murine EpoR, is replaced with a Cys moiety (EpoR(R129C)) rendering this receptor constitutively active. Over-expression of EpoR(R129C) permits cytokine-independent growth of BaF3 cells and renders these cells tumourigenic in nude mice (Yoshimura et al (1990) Nature 348:647). Mechanistically it is thought that the R129C mutation within the murine EpoR renders it constitutively active by allowing the receptors to dimerize. Similarly, mutation of either Glu132 or Glu133 (position is relative to the putative amino terminus at residue 25) to a Cys residue within the extracellular domain of the murine EpoR also results in a constitutively active EpoR (Watowich et al (1994) Mol Cell Biol 14:3535). A truncated murine EpoR containing an R129C mutation has also been identified and is constitutively active (Yoshimura et al (1990) Nature 348:647).
In vivo studies whereby the env gene of the spleen focus-forming virus is replaced by EpoR(R129C) have demonstrated that the modified virus induces transient thrombocytosis and erythrocytosis in infected mice and that the EpoR(R129C) stimulates the proliferation of committed megakaryocytic and erythroid progenitors as well as nonerythroid multipotent progenitors (Longmore et al (1994) Mol Cell Biol 14:2266-2277). Eight different multiphenotypic immortal cell lines, including primitive erythroid, lymphoid and monocytic cells, were isolated from the infected mice. All of these lines contained a mutant form of the p53 gene. These data suggest that a constitutively active form of the murine EpoR can induce proliferation and lead to transformation of nonerythroid as well as very immature erythroid progenitor cells when accompanied by a mutation of the p53 gene. A constitutively active form of the murine EpoR has been transfected into fetal liver cells (Pharr et al (1993) Proc Natl Acad Sci 90:938) and into pluripotent progenitors cells (Pharr et al (1994) Proc Natl Acad Sci 91:7482). When transfected into fetal liver cells, the activated EpoR eliminated the Epo requirement of CFU-E to form erythrocytes after 2-5 days in culture; however, this receptor did not support BFU-E development in the absence of Epo. The effect of Epo on the development of BFU-E was not investigated. Introduction of this constitutively active murine EpoR into pluripotent progenitors supported erythroid development in mixed colonies (GEMM) in the absence of Epo; however, its effect on committed erythroid progenitors (BFU-E or CFU-E) was not reported. Expression of this receptor did not alter the developmental potential of the infected pluripotent progenitors.
Several mutations within the intracellular domain of the human EpoR have been described for patients with primary familial and congenital polycythemia (PFCP). This disorder is characterized by elevated red blood cell mass and low serum Epo levels. Six of eight mutations result in truncation of the EpoR rendering them hypersensitive to Epo (Kralovics et al (1997) Blood 90:2057-2061). A constitutively active form of the human EpoR has not been described to date.
In Vitro Erythroid Cell Expansion and Differentiation
Traditionally, media for mammalian cell culture included a certain percentage of fetal bovine serum (FBS). However, serum contains undefined components, which may vary from batch to batch and may also be a source of contamination and growth factors. These materials may complicate the identification of multiple interactions that control proliferation and differentiation. To address these issues, a variety of serum-free fully defined media for the culture of hematopoietic cells has been described. Many of these formulations are similar in that they all include a basal medium such as IMDM, albumin, hormones and a source of fatty acids such as lipids and low density lipoproteins. As early as 1980, Iscove et al (Exp. Cell Res. 126:121-126) reported the complete replacement of serum with albumin, transferrin, iron, unsaturated fatty acids, lecithin and cholesterol in cultures of primary erythroid precursors. Recently, Polini et al (1997, Hematol. Cell Ther. 39:49-58) used a serum-free medium consisting of IMDM supplemented with BSA, human transferrin and insulin, soybean lecithin, cholesterol, hydrocortisone, inositol, folic acid and xcex1-thioglycerol to maintain human hematopoietic stem cells for an extended period of time ex vivo.
Several serum-free media have also been described for the ex vivo expansion and differentiation of erythrocytes from hematopoietic stem cells. Previously, Fibach et al (1991, Intl. J. Cell Cloning 9:57-64) described a two-stage culture method for the generation of erythrocytes from whole blood LDMNC. In the first stage, cells were cultured in the presence of FBS, xe2x80x9c5637 conditioned mediumxe2x80x9d and cyclosporin A and in the second stage, cells were cultured in the presence of Epo and FBS at reduced O2 tensions. While this method yielded more normoblasts/erythocytes than were contained in BFU-E colonies seeded in methylcellulose on day one, it employed both FBS and a conditioned medium. Both these components contain unknown substances which may be potential sources of contamination making this method undesirable.
Lansdorp and Dragowska (1992, J. Exp. Med. 175:1501-1509) and Malik et al (1998, Blood 91:2664-2671) reported the generation of erythrocytes from CD34+cells in serum-free medium. In both cases, CD34+cells were seeded in serum-free medium and serially passaged for the duration of the culture. Cells were reported to progress through erythropoiesis with the majority of the cells acquiring erythroid markers such as glycophorin A (GlyA) and the morphology of erythroblasts. With the addition of 2% BSA, 10 ug/ml insulin, 200 ug/ml transferrin, 40 ug/ml LDL and 20 ng/ml IL-3, 50 ng/ml SCF, 3U/ml Epo and 10 ng/ml IL-6 to IMDM, Lansdorp and Dragowska obtained 105 and 106 fold expansion from two samples of CD34+cells. The serum free formulation of Malik et al was composed of IMDM with 1% BSA, 10xe2x88x926M hydrocortisone and 10 U/ml Epo, 1 pg/ml GM-CSF and 0.01 U/ml IL-3. Although the degree of expansion was not reported, at culture termination, 10% to 40% of the cells cultured in this medium were reported to be nearly fully differentiated reticulocytes.
This invention teaches a method for the large-scale serum-free expansion of primary erythroid cells from CD34+cells. This method results in greater than 10,000,000-fold expansion of CD34+ cells of which more than 95% are erythroid in nature. Cells cultured according to the method of the invention, including erythroid progenitors, can be efficiently transfected by means of electroporation. Two constitutively active forms of the human EpoR and a hypersensitive form of the human EpoR are also described and their utility in prolonging the lifespan of erythroid progenitors in culture is demonstrated.
A truncated (t439)(relative to the imitation Met residue) version of the human EpoR gene has been constructed which contains a point mutation (R154C) (relative to the imitation Met residue). Transfection of this EpoR (xe2x80x9cEpoR(t439; R154C) into a cytokine-dependent cell line supports cell population expansion in the absence of exogenous cytokines. Transfection of this construct into hematopoietic progenitor cells increases the expansion of BFU-E, as detected using colony-forming assays.
It is thus an object of the invention to provide a serum free defined medium substantially free of fatty acids and hydrocortisone comprising effective amounts of: serum albumin, insulin, transferrin, IL-3, SCF, and EPO.
It is a further object of the invention to provide a method of producing an expanded population of erythroid cells comprising culturing an initial population containing erythroid precursors in a serum free defined medium.
It is a further object of the invention to provide a method of producing a differentiated population of erythroid cells comprising culturing an initial population containing erythroid precursors in a serum free defined medium.
It is a further object of the invention to provide a method of producing a population of erythroid cells suitable for high efficiency non-viral transfection comprising culturing an initial population containing erythroid precursors in a serum free defined medium comprising serum albumin, insulin, transferrin, IL-3, SCF, and EPO.
It is a further object of the invention to provide a method of producing a population of erythroid cells suitable for high efficiency non-viral transfection comprising culturing an initial population containing erythroid precursors in a serum free defined medium comprising serum albumin, insulin, transferrin, IL-3, SCF, EPO, and a fatty acid source, and subsequently treating the cells for high efficiency non-viral transfection.
It is a further object of the invention to provide a method of producing a population of erythroid cells having a hemoglobin (Hb) content in excess of normal levels comprising culturing an initial population in a serum-free defined medium comprising serum albumin, insulin, transferrin, IL-3, SCF, and EPO and essentially free of fatty acid sources.
It is a further object of the invention to provide a population of erythroid cells suitable for high efficiency non-viral transfection.
It is a further object of the invention to provide a population of erythroid cells having a Hb content in excess of normal levels.
It is a further object of the invention to provide a method of transfecting erythroid cells by electroporation comprising:
(a) culturing an initial cell population in a serum free defined culture medium comprising effective amounts of serum albumin, insulin, transferrin, IL-3, SCF, Epo, and LDLs, and
(b) electroporating cells in the presence of suitable DNA
It is a further object of the invention to provide a constitutively active human EpoR.
It is a further object of the invention to provide a human EpoR truncated at amino acid 439.
It is a further object of the invention to provide a human EpoR having an Rxe2x86x92C mutation at amino acid residue 154.
It is a further object of the invention to provide a human EpoR having an Rxe2x86x92C mutation at amino acid residue 154 which is truncated at amino acid residue 439.
It is a further object of the invention to provide an erythroid cell expressing a human EpoR having an Rxe2x86x92C mutation at amino acid residue 154 which is truncated at amino acid residue 439.
It is a further object of the invention to provide a use of an effective amount of a suitable heme synthesis inhibitor in enhancing the expansion of a population of erythroid cells.
It is a further object of the invention to provide a use of an expanded population of erythroid cells in hematologic support.
It is a further object of the invention to provide a use of a differentiated population of erythroid cells in hematologic support.
It is a further object of the invention to provide a use of a population of erythroid cells suitable for high efficiency non-viral transfection in gene therapy.
It is a further object of the invention to provide a use of an expanded population of erythroid cells in in vitro viral replication.
It is a further object of the invention to provide a use of a differentiated population of erythroid cells in in vitro viral replication.
It is a further object of the invention to provide a use of an expanded population of erythroid cells in replicating parvovirus B19 in vitro.
It is a further object of the invention to provide a use of a differentiated population of erythroid cells in replicating parvovirus B19 in vitro.
It is a further object of the invention to provide a use of a hemoglobin source in enhancing Hb production in a population of erythroid cells expanded in the presence of a heme synthesis inhibitor.